Species

Description and significance

Granulobacter bethesdensis is a recently identified bacterial pathogen which was discovered in the excised lymph nodes of a chronic granulomatous disease patient. CGD is a rare immune disorder that leaves individuals susceptible to frequent and severe fungal and bacterial infections. This microbe is a gram negative, rod shaped bacteria thought to cause fever and lymphadenitis. Through a multilocas phylogenetic analysis based on the 16S rRNA gene, the internal transcribed spacer region and the RecA protein, this novel bacterium has been recognized as belonging to a new lineage within the Acetobacteraceae family. Bacteria belonging to the acetobacteraceae family have been classified as acetic acid bacteria, based on the characteristic of deriving energy from the oxidation of ethanol to acetic acid during respiration. Granulobacter bethesdensis was granted a separate genus classification due to its unique specificities including its standing as a methylotroph, its production of yellow pigment, and its weakness in producing acetic acid from ethanol. The acetobacteraceae family includes other types of bacteria that are very prevalent in the environment. Industrial uses of these bacteria include the production of vinegar and they are often encountered in the fermentation of wine. This particular bacterium, however, is the first in its family to have the ability to cause invasive human disease. The bacterium was appropriately named; taking into consideration the meaning of granulobacter, a rod that causes granules or granuloma formation, and recognizing the place of discovery, Bethesda, Md., the location of the National Institution of Health. (1) (2)

Since granulobacter bethesdensis was a previously unrecognized bacterium, it had to undergo many tests in order to prove clinical correlation. Several steps were taken to assure the pathogenicity of this new organism. First, the specific antibody response in the patient was measured. Once infected, the patient seroconverted from negative to high titer antibody. Additionally, the pathogenicity of the new bacterium was proven by applying Koch's postulates. After the bacterium was identified and isolated from the patient's lymph nodes, it was introduced into mouse models of CGD and wild type mice. Infection of the bacteria proved to cause disease in the mice as well. Both the CGD and wild type mice developed the antibody but the human immunopathology was strongly replicated in the CGD mouse. The CGD and wild type mice showed pathologic changes in their lymph nodes; however, it was much milder in the latter. Then the organism was once again isolated by removing it from the pathologic lesions in the mice. This process solidified its association with the disease.(1) (2)

Genome structure

The genome of Granulobacter bethesdensis is 2,708,355 nucleotides long with a guanine-cytosine content of 59.07%. In total, the genome of the organism is made up of 2498 genes; 2437 of these genes coding for proteins. 61 of these genes are RNA genes, 3 are ribosomal RNA genes and 52 are tRNA genes. Granulobacter bethesdensis has a circular genome with a length of 2.7 Mbp.(6)

Correct identification and classification of the bacterium required genetic sequencing. The bacteria had been found and isolated from three different patients. The 16S rRNA genes from each of these isolations were sequenced, the sequences analyzed, and then compared to each other to prove identicality. Then these gene sequences were compared to the 16S rRNA gene sequences available in the GenBank. No identical matches were found but this analysis uncovered the similarities between the newly found organism and other organisms in the Acetobacteraceae family. Genetically, granulobacter bethesdensis's closest match was gluconacetobacter sacchari with 95.7% similarity. However, in the end, the 16S rRNA gene sequence of strain CGDNIH1 warranted a separate genus level status. Genome sequencing was completed on September 24th, 2006 at the Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease.(2) (1)

Cell structure and metabolism

The structure of granulobacter bethesdensis is described to be rod shaped, but more specifically coccabacillus. Coccabacillus refers to a subset of rod shaped bacteria that are characteristically shorter and wider, seeming to resemble cocci. This organism is considered to be non motile and has no endospores. Granulobacter bethesdensis is a gram negative bacterium, distinguished by its inner and outer membrane. Examination of the microbe revealed that its two major fatty acids were a straight chain unsaturated acid and C16:0. (2) (5)

During the initial investigation of the species, it was found to be catalase positive, oxidase negative and urease variable. Catalase is a common enzyme which is found in living organisms; the function of this enzyme is to catalyze the decomposition of hydrogen peroxide into water and oxygen. Catalase positivity is determined through the presence of bubbles or froth when the colony comes in contact with hydrogen peroxide on the microscope slide. Similarly, oxidase and urease are enzymes as well. The cause of urease variability comes from the fact that it was positive in only two out of the three isolations. (2)

Granulobacter bethesdensis is classified as an aerobic bacterium, meaning that it uses oxygen to oxidize substrates for obtaining energy during respiration. The bacterium is able to oxidize lactate and, more weakly, acetate. It produces little acetic acid from ethanol which differentiates it from other acetic acid bacteria which are known for their ability to oxidize alcohols and sugars, specifically acetic acid from ethanol. Granulobacter bethesdensis does produce acid from glucose and ethanol. It was has been seen that the organism grows on glutamate and mannitol agar and forms colonies that are yellow pigmented. It was also documented that the organism preferred high concentrations of glucose for growth. Granulobacter bethesdensis is a methylotroph so it has the capability to use methanol as a sole carbon source.(2)

Ecology

Granulobacter bethesdensis was isolated from cervical and supraclavicular lymph nodes of a patient who had been diagnosed with chronic granulomatous disease. The organism grew at an optimum pH of 5-6.5 and at an optimal temperature of 35-37 degrees Celsius, making it mesophilic. Most other acetic acid bacteria grow at an optimal temperature of approximately 30 degrees. The significance of this species growing at a higher temperature can be attributed to the fact that 37 degrees is a common optimal temperature for many pathogenic mesophiles because 37 degrees Celsius doubles as normal human body temperature (98 degrees Fahrenheit). The microbe prefers to grow in environments with high glucose concentrations. Granulobacter bethesdensis is non halophilic organism meaning that it does not thrive in environments having high saline concentrations. Acetobacteraceae in general are considered ubiquitous environmental organisms.(1) (2)

Pathology

Granulobacter bethesdensis was discovered during the investigation of a patient with CGD. Chronic granulomatous disease is a hereditary disease of the immune system. This disease affects the phagocyte NADPH oxidase system which leads to defective production of superoxide and hydrogen peroxide. These reactive oxygen compounds are used to kill certain ingested pathogens. Thus the absence of these of these compounds impairs the killing of microbes and consequently weakens the immune system. Patients diagnosed with CGD suffer from infections by catalase producing organisms and develop tissue granulomas. Examples of organisms associated with this disease include members of the genera Staphyloccocus, Serratia, Burkholderia, Nocardia and Aspergillus. (1)

Granulobacter bethesdensis was added to this list when it was isolated from a CGD patient in August 2003 (patient 1). The organism was found in two other CGD patients in December 2005 and January 2006. This was the first bacteria in its family found to cause human disease. Patient 1 had lived with his condition for a number of years but was hospitalized in April 2003 for fever and chills. A computed tomography scan of his chest showed two calcified granulomata in the right lower lobe. A month later a biopsy of the carinal lymph nodes showed caseating granulomatous inflammation. Five months after the initial symptoms the patient developed left supraclavicular lymphadenopathy. Due to the lack of microbiologic diagnosis, the left cervical and supraclavicular lymph nodes were removed. The patient had produced antibodies in response to the microbe but it seemed that the antibody production was not able to control the infection. (1)

It was at this point that the organism was isolated and discovered. After subsequent research, it was concluded that this new bacterium caused fever, lymphadenitis and weight loss in the patient but it did not prove to be fatal, neither in the patients nor in the mice. The mice used in the challenge studies were followed for up to four months after infection and all the mice cleared the infection spontaneously over time.(1)

Application to Biotechnology

Acetic acid bacteria derive energy from the aerobic oxidation of ethanol to acetic acid. The have been isolated from fruits, fermented foods, plants, soil and water and are important in the food and biotechnology industry. However, granulobacter bethesdensis has no known applications to biotechnology as of yet.(2)

Current Research

The discovery of this organism has been ensued by intense research of the organism itself and its disease causing abilities. Fifty percent of infections in CGD patients have not been able to be diagnosed. So, in the past, these patients were treated empirically with surgery, a variety of drugs, or in some cases, the infections were never successfully treated. The discovery of the existence of granulobacter bethesdensis may help in better understanding CGD and the infections surrounding it so that more helpful therapies can be determined. Patients with CGD are understood to be susceptible to infections caused by catalase producing organisms. This occurs because these organisms scavenge their own hydrogen peroxide and in doing so they deprive the neutrophil of the opportunity to use oxidative metabolites against them. But, there are many catalase producing organisms that do not cause disease in CGD. This infers that there are unknown mechanisms, other that catalase, which confers pathogenicity on organisms that infect patients with CGD. The discovery of this new bacterial pathogen will hopefully contribute to the furthered understanding of these mechanisms.(1) (2)

Current research will also focus on the many unknown qualities of the organism and its infection. The organism was able to be repeatedly isolated from the patients’ lymph nodes and this leads to uncertainty about whether the patient had relapsed, was re-infected, or if there is an unidentified environmental reservoir.(2)

The clinical epidemiology of this organism also will have to be examined. The difficulty in culturing this organism from pathologic specimens and the appearance of antibodies even in healthy people suggests that there have been previous, unacknowledged, encounters with this organism. David Greenberg, M.D., the lead author on the study, has suggested that further investigation will be needed in three areas. First, the immune response to the new bacteria in CGD patients will have to be contrasted to the immune response to granulobacter bethesdensis in healthy people. Secondly the process by which the microbe causes infection in people with CGD needs to be better understood. Lastly, it should be determined whether the organism holds any significance for the population at large.(2) (1)